Efficient Transmission of Pandemic H1N1 Influenza Viruses with High-Level Oseltamivir Resistance

Abstract

The limited availability of approved influenza virus antivirals highlights the importance of studying the fitness and transmissibility of drug-resistant viruses. S247N is a novel, naturally occurring N1 neuraminidase mutation that reduces oseltamivir sensitivity and greatly potentiates oseltamivir resistance in the context of the H275Y mutation. Here we show that highly oseltamivir-resistant viruses containing both the S247N and H275Y mutations transmit efficiently in the guinea pig transmission model.

TEXT

Currently circulating pandemic H1N1 (pH1N1) viruses are sensitive to neuraminidase (NA) inhibitors, including oseltamivir, but the speed with which a well-described oseltamivir resistance mutation, H275Y, achieved fixation among seasonal H1N1 (sH1N1) viruses between 2007 and 2009 argues for a timely assessment of the fitness and transmissibility of naturally occurring drug-resistant strains (13, 23). The ability of sH1N1 H275Y viruses to transmit is especially alarming because viruses prior to 2007 were attenuated by the H275Y mutation (1, 16), suggesting that compensatory mutations acquired by the virus, and more specifically the NA (N. M. Bouvier, S. Rahmat, and N. Pica, unpublished data), increased transmissibility (1–3, 5, 9, 16, 18, 27).

Between December 2010 and March 2011, a cluster of pH1N1 community isolates from Australia and Singapore was found to encode a novel N1 resistance mutation, S247N, which, when found in combination with H275Y, resulted in a highly oseltamivir-resistant virus for which the median inhibitory concentration (IC₅₀) was almost 6,000-fold greater than that for wild-type isolates (15). Because pH1N1 H275Y viruses are as pathogenic as wild-type viruses and transmit well under most conditions in animal models (7, 11, 17, 24, 28, 32), we sought to determine if highly oseltamivir-resistant strains encoding both the S247N and the H275Y NA mutations could also efficiently transmit by respiratory droplets, including aerosols and droplets of all sizes.

Cal09 virus containing the S247N and H275Y NA mutations transmits less efficiently than wild-type virus.

Pandemic A/California/04/2009 (H1N1)-based viruses (8, 10, 28) with the NA mutations S247N (i.e., Cal09-S247N) and H275Y (Cal09-H275Y) along with a combination mutation (Cal09-S247N+H275Y) were rescued. A wild-type virus (Cal09-wt) was rescued for comparison, and hemagglutinin (HA) and NA segments for all viruses were verified by sequencing. Using a 2′-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (MUNANA)-based assay, we confirmed that S247N independently increases oseltamivir resistance, which is further enhanced 680-fold by the addition of the H275Y mutation (Table 1). In Madin-Darby canine kidney (MDCK) cells infected at a multiplicity of infection (MOI) of 0.01, the multicycle growth kinetics of the three mutant viruses were similar to that of recombinant Cal09-wt, demonstrating their in vitro fitness (Fig. 1A).

Table 1.

Enzymatic properties of the neuraminidases expressed by recombinant viruses

Recombinant virus	Oseltamivir		Zanamivir		Km (μM)	Transmission efficiency (%)
	Mean IC50 (nM)	Fold changeb	Mean IC50 (nM)	Fold changeb
Cal09-wt	1.0		0.62		53	100a
Cal09-H275Y	280	280	0.49	0.80	180	88a
Cal09-S247N	3.7	3.7	1.3	2.1	45	88
Cal09-H275Y+S247N	2,500	2,500	2.0	3.2	73	50
Cal09-Aus	2.7	2.7	0.66	1.1	150	88
Cal09-Aus H275Y	2,000	2,000	1.2	2.0	390	88

^aHistorical data from reference 28 (2010).

^bFold change relative to the wild type.

Fig 1.

Cal09 NA and Australian NA mutant viruses grow with multicycle growth kinetics similar to that of wild-type Cal09 virus in MDCK cells. (A) Growth of highly drug-resistant, recombinant Cal09-S247N+H275Y in MDCK cells was similar to growth of viruses mutated at either NA S247N or NA H275Y along with wild-type virus. (B) Viruses with Australian clade NAs (V106I, S247N, N248D, and S299A with or without H275Y) in the Cal09 background grew similarly to Cal09-wt and Cal09-H275Y viruses in MDCK cells. Results are representative of 2 independent trials in triplicate. Error bars represent standard errors.

To evaluate the transmissibility of Cal09-S247N and Cal09-S247N+H275Y, four Hartley guinea pigs (Charles River Laboratories) were inoculated with 1,000 PFU of each virus and, 24 h later, transferred to wire-sided cages in an environmental chamber (Caron 6030) with four naïve guinea pigs. Each guinea pig was housed separately in an individual wire-sided cage, which allows air to flow freely among cages but prevents the animals from coming into direct contact. Inoculated and naïve animals were placed in pairs, each on a separate shelf in the environmental chamber (as shown in Fig. 1 in reference 19). Nasal washes were performed every other day, and titers were determined as previously described (4, 19, 20, 29, 30). The Cal09-wt virus was previously shown to transmit at an efficiency of 100% under conditions identical to those in the current experiments (28). Interestingly, the Cal09-S247N virus transmitted to 88% of naïve animals (7 of 8); however, Cal09-S247N+H275Y transmitted to only 50% of naïve animals (4 of 8) (Fig. 2A and B). While the transmission efficiency of Cal09-S247N (88%) is similar to that shown previously for Cal09-wt (100%) and Cal09-H275Y (88%) (28), the double mutant Cal09-S247N+H275Y showed a statistically significant decrease in transmission efficiency relative to Cal09-wt (P < 0.05 by Fisher's exact test), suggesting that transmission is attenuated as a consequence of its high oseltamivir resistance phenotype. Still, it is significant that a virus that can achieve such high levels of drug resistance is able to transmit in our model, as not all drug-resistant viruses transmit by respiratory droplets among guinea pigs, even when they replicate efficiently in vitro (4).

Fig 2.

Transmission efficiencies for drug-resistant viruses with the S247N mutation or both the S247N and H275Y mutations in the guinea pig model. Cal09-S247N transmitted to 88% of exposed, naïve guinea pigs (A), while the double mutant with increased drug resistance, Cal09-S247N+H275Y, transmitted to only 50% of exposed animals (B). (C and D) Viruses with the Australian clade NA (V106I, S247N, N248D, and S299A with or without H275Y) in the Cal09 background efficiently spread to exposed, naïve guinea pigs at an efficiency of 88%. Transmission efficiencies of each virus were determined in two independent experiments, with 4 exposed, naïve guinea pigs per experiment.

Cal09-wt with the Australian clade NA (V106I, S247N, N248D, and S299A) transmits efficiently with and without the H275Y mutation.

The S247N-encoding viruses identified by Hurt et al. (15) also contained three consensus amino acid substitutions in the NA (V106I, N248D, and S299A) relative to the Cal09-wt used in our initial transmission studies. These mutations could potentially alter NA protein structure, expression, or enzymatic activity in the context of the S247N mutation or the combination of S247N with H275Y (2). To evaluate any effect on transmission or growth kinetics, we rescued viruses in the Cal09 background that express the “Australian” NA (V106I, S247N, N248D, and S299A, relative to the Cal09 NA) (Cal09-Aus) or the Australian NA with the additional oseltamivir-resistance mutation H275Y (Cal09-Aus H275Y). While these viruses may differ from the original Australian and Singaporean isolates in the other seven segments, they express the same drug-resistant NA as the Australian viruses. In multicycle growth on MDCK cells, both Cal09-Aus and Cal09-Aus H275Y grew with kinetics similar to that of Cal09-wt (Fig. 1B). Surprisingly, though, both viruses transmitted with 88% efficiency, to 7 of 8 naïve animals (Fig. 2C and D). This result contrasts with our earlier observation that Cal09-S247N+H275Y displayed deficient transmissibility compared to Cal09-wt. Notably, the area under the exposed animals' nasal wash curves (AUCs) for the four different viruses revealed no statistically significant differences that might suggest differences in transmission kinetics (P > 0.72 by Kruskal-Wallis test). To evaluate whether differences in enzymatic activity might be playing a role in the transmission phenotype, Michaelis-Menten kinetics were evaluated using 10^5.7 PFU of each virus incubated with MUNANA (final concentrations between 0 and 1,388 μM) for 40 min (7, 26, 27). There was no clear correlation between the K_m and transmission phenotype; however, in each case, the H275Y mutation decreased affinity for the MUNANA substrate compared to the relevant parental virus (Table 1). Overall, slight differences in transmissibility between Cal09-Aus and Cal09-Aus H275Y, which may be relevant on a population level, may not be detectable by the sensitivity of our assay.

Drug-resistant viruses that transmit efficiently in animal models may not become widely prevalent in humans. Cal09-H275Y transmitted efficiently in both guinea pigs and ferrets (even when coinoculated with wild-type virus [28]), even though oseltamivir-resistant Cal09-like viruses were not widely observed in the human reservoir. However, scattered transmission events of pH1N1 H275Y viruses were recorded in 2009 (6, 12, 21, 22, 25, 31), and more recently, pH1N1 viruses with the H275Y mutation (but without S247N) were identified in up to 16% of samples near Newcastle, New South Wales, Australia, suggestive of community transmission (14). Thus, it is of concern that Cal09-Aus H275Y, for which the oseltamivir IC₅₀ is approximately 2,000-fold higher than that for the wild-type virus, is able to transmit efficiently in the guinea pig model. Additionally, the enhancement in transmissibility observed with just three amino acid substitutions to the Cal09-S247N NA adds credence to the hypothesis that compensatory mutations in the NA enable the transmissibility of drug-resistant viruses. These results support continued active surveillance for S247N viruses that may acquire the H275Y mutation, as well as the use of animal models for rapid assessment of the transmissibility of other naturally occurring drug-resistant strains of influenza virus.